Flower petals perform the role of attracting pollinators such as insects and birds, which transport plant pollen, and therefore flower colors, shapes, patterns and odors have evolved in tandem with pollinators (Honda, T. et al., Gendai Kagaku, May, 25-32 (1998)). Probably as a result of this, it is rare for a single species of flower to exhibit several different colors, and for example, rose or carnation varieties exhibiting violet to blue colors do not exist, while iris or gentian varieties exhibiting bright red colors do not exist. Because flower color is the most important aspect of petals for purposes of appreciation as well, flowers of different colors have traditionally been bred by crossbreeding. The rose, known as the “queen of flowers” and having high commercial value, has also been crossbred throughout the world.
For example, the current yellow rose cultivar was created by crossbreeding of Rosa foetida, originating from western Asia, with a non-yellow rose variety. However, because flower color is determined by the genetic capacity of the plant, there has been a limit to the flower colors that can currently be produced in cross-bred strains whose available genetic sources are restricted (Tanaka et al. Plant Cell Physiol. 39, 1119-1126, 1998; Mol et al. Curr. Opinion Biotechnol. 10, 198-201 1999). Among these, the cultivation of blue roses has been thought impossible and has been considered the “holy grail” of colors (Oba, H., “Bara no Tanjo”, 1997, Chukoshinsho; Suzuki, M., “Shokubutsu Bio no Mahou: Aoi Bara mo Yume dewanakuhatta”, 1990, Kodansha Bluebacks; Saisho, H., “Aoi Bara”, 2001, Shogakkan).
Although “blue rose” varieties currently exist, these are actually pale violet roses. The first improved variety of “blue rose” by crossbreeding is said to have been the light-violet shaded grey-colored “Grey Pearl” created in 1945. The light-violet pink-colored “Staring Silver” was later created in 1957, and these varieties were crossed to produce several pale violet roses such as “Blue Moon” (1964) and “Madam Violet” (1981). These pale violet roses and other roses were then utilized in further breeding to create light-grey-colored roses such as “Seiryu” (1992) and “Blue Heaven” (2002), which were hailed as new types of “blue roses”.
However, these flower colors are not actually blue but merely greyish-dull pink, and despite many years of breeding efforts, there is still no example of a truly “blue” rose. In horticultural industry, the group of colors from violet to blue is generally considered “blue” according to the RHSCC (The Royal Horticultural Society Colour Chart). It is an aim of the present invention to create rose plants having flower colors falling within the “violet group”, “violet-blue” group and “blue group” according to the Royal Horticultural Society Colour Chart.
Flower colors derive mainly from the three compound groups of anthocyanins, carotenoids and betalains, but it is the anthocyanins, having the widest absorption wavelength range (from orange to blue), that are responsible for blue color. Anthocyanins belong to the flavonoid family and are biosynthesized by the metabolic pathway shown in FIG. 1. Anthocyanins are normally localized in the vacuoles of epithelial cells. The color shade of anthocyanins (i.e. flower color) depends largely on the structure of the anthocyanins, with more numerous hydroxyl groups on the B ring resulting in a bluer color. Hydroxylation of the B ring is catalyzed by flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Absence of F3′H and F3′5′H activity leads to synthesis of pelargonidin (orange to red colors), presence of F3′H activity leads to synthesis of cyanidin (red to rouge colors) and presence of F3′5′H activity leads to synthesis of delphinidin (violet color).
These anthocyanidins are modified with sugars and acyl groups to produce an assortment of anthocyanins. Generally speaking, a larger number of modifying aromatic acyl groups correlates to bluer anthocyanins. Anthocyanins also produce quite different colors depending on the vacuole pH and the copresent flavonols and flavones or metal ions (Saito, N., Tanpakushitsu Kakusan Kouso, 47 202-209, 2002; Broullard and Dangles, In the flavonoids: Advances in Research since 1986 (Ed. by Harborne) Capmann and Hall, London pp. 565-588; Tanaka et al. Plant Cell Physiol. 39 1119-1126, 1998; Mol et al., Trends in Plant Science 3, 212-217, 1998; Mol et al., Curr. Opinion Biotechnol. 10, 198-201 1999).
Rose flower petal anthocyanins are derivatives of pelargonidin, cyanidin and peonidin, whereas no delphinidin derivatives are known (Biolley and May, J. Experimental Botany, 44, 1725-1734 1993; Mikanagi Y., Saito N., Yokoi M. and Tatsuzawa F. (2000) Biochem. Systematics Ecol. 28:887-902). This is considered to be the main reason for the lack of blue roses. Existing roses have been created by crossbreeding of crossable related rose species (R. multiflora, R. chinensis, R. gigantean, R. moschata, R. gallica, R. whichuraiana, R. foetida, etc.).
The fact that no blue rose has been achieved in spite of repeated efforts at crossbreeding is attributed to the lack of delphinidin production ability by rose-related varieties. Production of delphinidin in rose petals would require expression of F3′5′H in the petals as mentioned above, but F3′5′H is believed to be non-expressed in the petals of rose and rose-related varieties. Thus, it is likely impossible to obtain a blue rose by accumulating delphinidin in the petals through crossbreeding. It is known that trace amounts of the blue pigment rosacyanin are found in rose petals and its chemical structure has been determined (Japanese Unexamined Patent Publication No. 2002-201372), but no reports are known regarding augmentation of rosacyanin to create a blue rose, and no findings have been published on the rosacyanin biosynthesis pathway or the relevant enzymes or genes.
Examples of blue or violet colors produced by biological organisms also include indigo plant-produced indigo (for example, Appl. Microbiol. Biotechnol. February 2003, 60(6):720-5) and microbially-produced violacein (J. Mol. Microbiol. Biotechnol. October 2000 2 (4):513-9; Org. Lett., Vol. 3, No. 13, 2001, 1981-1984), and their derivation from tryptophan and their biosynthetic pathways have been studied.
Blue pigments based on gardenia fruit-derived iridoid compounds (S. Fujikawa, Y. Fukui, K. Koga, T. Iwashita, H. Komura, K. Nomoto, (1987) Structure of genipocyanin G1, a spontaneous reaction product between genipin and glycine. Tetrahedron Lett. 28 (40), 4699-700; S. Fujikawa, Y. Fukui, K. Koga, J. Kumada, (1987), Brilliant skyblue pigment formation from gardenia fruits, J. Ferment. Technol. 65 (4), 419-24) and lichen-derived azulenes (Wako Pure Chemical Industries Co., Ltd.) are also known, but no reports are known of expressing these in plant flower petals to produce blue-colored flowers.
It has been expected that a blue rose could be created by transferring the F3′5′H gene expressed by other plants into rose and expressing it in rose petals (Saisho, H., “Aoi Bara”, 2001, Shogakkan). The F3′5′H gene has been obtained from several plants including petunia, gentian and Eustoma russellianum (Holton et al. Nature 366, 276-279, 1993; Tanaka et al. Plan Cell Physiol. 37, 711-716 1996; WO93/18155). There are also reports of transformed varieties of rose (for example, Firoozababy et al. Bio/Technology 12:609-613 (1994); U.S. Pat. No. 5,480,789; U.S. Pat. No. 5,792,927; EP 536,327 A1; US 20010007157 A1).
Actual transfer of the petunia F3′5′H gene into rose has also been reported (WO93/18155, WO94/28140).
However, it has not been possible to obtain a blue rose, and it is believed that obtaining a blue rose will require a modification which alters the metabolism of flower pigments suited for rose.
On the other hand, it has been confirmed that transfer of the F3′5′H gene into red carnation, which produces pelargonidin instead of delphinidin, leads to accumulation of both pelargonidin and delphinidin, but that the flower color is only altered to a slightly purplish red (WO94/28140). This result suggests that it is not possible to obtain a “blue” carnation simply by expression of F3′5′H, and that it is necessary to inhibit the metabolic pathway to endogenous synthesis of pelargonidin by carnation.
In order to avoid competition with the carnation endogenous metabolic pathway (reduction of dihydrokaempferol (DHK) by dihydroflavonol reductase (DFR)), a variety lacking DFR was selected from among white carnations. The F3′5′H gene and petunia DFR (which is known to efficiently reduce dihydromyricetin (DHM) without reducing DHK) gene were transferred into carnation. This resulted in one case of successfully obtaining a recombinant carnation with a delphinidin content of about 100% and a blue-violet flower color previously not found in carnation (Tanpakushitsu Kakusan Kouso, Vol. 47, No. 3, p 225, 2002). Thus, further modification was necessary to realize a blue carnation flower, in addition to accumulating delphinidin by expression of the F3′5′H gene.
DFR has already been cloned from several plants (petunia, tobacco, rose, Torenia, snapdragon, transvaal daisy, orchid, barley, corn, etc.) (Meyer et al., Nature 330, 677-678, 1987; Helariutta et al., Plant Mol. Biol. 22, 183-193 1993; Tanaka et al., Plant Cell Physiol. 36, 1023-1031; Johnson et al., Plant J. 19, 81-85, 1999). Substrate specificity of the DFR gene differs depending on the plant variety, and it is known that the petunia, tobacco and orchid DFR genes cannot reduce DHK, whereas the petunia DFR gene most efficiently reduces DHM among the dihydroflavonols (Forkmann et al., Z. Naturforsch. 42c, 1146-1148, 1987; Johnson et al. Plant J. 19, 81-85, 1999). Nevertheless, no cases have been reported for expression of these DFR genes in rose.
As a means of avoiding competition with the endogenous metabolic pathway or between the enzyme and the exogenous gene-derived enzyme such as F3′5′H, as mentioned above, the gene may be transferred into a variety lacking the gene. Also, it is known that expression of the target gene can be artificially inhibited by deletion methods involving homologous recombination of the target gene, but because of the low frequency of homologous recombination and the limited number of suitable plant varieties, this has not been implemented in practice (for example, Nat. Biotechnol. 2002, 20:1030-4).
Inhibition methods on the transcription level include the antisense method using antisense RNA transcripts for mRNA of the target gene (van der Krol et al., Nature 333, 866-869, 1988), the sense (cosuppression) method using transcripts of RNA equivalent to mRNA of the target gene (Napoli et al., Plant Cell 2, 279-289, 1990) and a method of using duplex RNA transcripts corresponding to mRNA of the target gene (RNAi method; Waterhouse et al., Pro. Natl. Acad. Sci. USA 95, 13959-13964, 1998).
Numerous successful examples of these three methods have been published. For rose, cosuppression of chalcone synthase (CHS) gene which is necessary for synthesis of anthocyanins was reported to successfully alter flower color from red to pink (Gutterson HortScience 30:964-966 1995), but this CHS suppression is incomplete and therefore it has not been possible to totally suppress anthocyanin synthesis to obtain a white flower stock.    Patent document 1: Japanese Unexamined Patent Publication No. 2002-201372    Patent document 2: WO93/18155    Patent document 3: U.S. Pat. No. 5,480,789    Patent document 4: U.S. Pat. No. 5,792,927    Patent document 5: EP 536 327 A1    Patent document 6: US 20010007157 A1    Patent document 7: WO94/28140    Non-patent document 1: Honda T. et al. Gendai Kagaku, May, 25-32 (1998)    Non-patent document 2: Tanaka et al. Plant Cell Physiol. 39, 1119-1126, 1998    Non-patent document 3: Mol et al. Curr. Opinion Biotechnol. 10, 198-201 1999    Non-patent, document 4: Oba, H., “Bara no Tanjo”, 1997, Chukoshinsho    Non-patent document Suzuki, M., “Shokubutsu Bio no Mahou: Aoi Bara mo Yume dewanakunatta”, 1990, Kodansha Bluebacks    Non-patent document 6: Saisho, H., “Aoi Bara”, 2001, Shogakkan    Non-patent document 7: Saito, N., Tanpakushitsu Kakusan Kouso, 47 202-209, 2002    Non-patent document 8: Broullard et al. In the flavonoids: Advances in Research since 1986 (Ed by Harborne) Capmann and Hall, London pp 565-588    Non-patent document 9: Tanaka et al. Plant Cell Physiol. 39 1119-1126, 1998    Non-patent document 10: Mol et al, Trends in Plant Science 3, 212-217 1998    Non-patent document 11: Mol et al. Curr. Opinion Biotechnol. 10, 198-201 1999    Non-patent document 12: Biolley and May, J. Experimental Botany, 44, 1725-1734 1993    Non-patent document 13: Mikanagi Y, et al. (2000) Biochem Systematics Ecol. 28:887-902    Non-patent document 14: Appl. Microbiol. Biotechnol. 2003 February; 60(6):720-5    Non-patent document 15: J. Mol. Microbiol. Biotechnol. 2000 October; 2 (4): 513-9    Non-patent document 16: Org. Lett., Vol. 3, No. 13, 2001, 1981-1984    Non-patent document 17: S. Fujikawa, et al. (1987) Tetrahedron Lett. 28 (40), 4699-700    Non-patent document 18: S. Fujikawa, et al. (1987) J. Ferment. Technol. 65 (4), 419-24    Non-patent document 19: Holton et al. Nature 366, 276-279, 1993    Non-patent document 20: Tanaka et al. Plant Cell Physiol. 37, 711-716 1996    Non-patent document 21: Firoozababy et al. Bio/Technology 12:609-613 (1994)    Non-patent document 22: Tanpakushitsu Kakusan Kouso, Vol. 47, No. 3, p 225, 2002    Non-patent document 23: Meyer et al. Nature 330, 677-678, 1987    Non-patent document 24: Helariutta et al. Plant Mol. Biol. 22 183-193 1993    Non-patent document 25: Tanaka et al. Plant Cell Physiol. 36, 1023-1031    Non-patent document 26: Johnson et al. Plant J. 19, 81-85, 1999    Non-patent document 27: Forkmann et al. Z. Naturforsch. 42c, 1146-1148, 1987    Non-patent document 28: Nat Biotechnol 2002, 20:1030-4    Non-patent document 29: van der Krol et al. Nature 333, 866-869, 1988    Non-patent document 30: Napoli et al. Plant Cell 2, 279-289, 1990    Non-patent document 31: Waterhouse et al. Pro. Natl. Acad. Sci. USA 95, 13959-13964 1998    Non-patent document 32: Gutterson HortScience 30:964-966 1995
Non-patent document 33: Suzuki, S., “Bara, Hanazufu”, Shogakkann, p. 256-260, 1990